Project Summary The origins of childhood obesity and subsequent poor metabolic health may begin in utero. The Hyperglycemia and Adverse Pregnancy Outcome Follow Up Study (HAPO FUS) provides substantial evidence that maternal hyperglycemia and BMI during pregnancy are strongly associated with obesity in their 10-14 year old children. However, a knowledge gap exists as mechanisms mediating the pathway between an adverse uterine environment and childhood obesity development have not been elucidated. Epigenetic DNA alterations triggered by an adverse uterine environment is a possible mechanism underlying associations between maternal hyperglycemia/obesity and childhood obesity. We hypothesize that aberrant offspring DNA methylation occurs in response to an adverse intrauterine environment, characterized by maternal hyperglycemia and/or obesity. Differential DNA methylation (DNAm) may affect metabolically-important genes contributing to fetal programming of adiposity, and higher rates of childhood obesity. To investigate these important questions, we will take advantage of the HAPO FUS cohort on whom we have detailed information about the uterine environment, direct measurements of newborn and childhood adiposity, and existing genetic data. Using stored cord blood and childhood DNA of 3243 HAPO FUS participants, the goals of this project are to conduct DNA methylation studies utilizing the MethylationEPIC 850K BeadChip (Illumina Infinium). In Aim 1, we will investigate cord blood DNAm in select candidate genes proposed to play a critical role linking maternal hyperglycemia/ BMI to newborn and childhood adiposity outcomes in HAPO FUS. Potential methylation-related mechanisms underlying these associations will be identified using mediation analysis. In Aim 2, we will integrate cord blood DNAm profile and existing SNP data from the HAPO genome-wide association study with mapping of methylation quantitative trait loci (mQTL) in order to elucidate the genetic architecture of CpG sites associated with offspring adiposity traits. We will then perform 2-step Mendelian randomization to identify causal CpG loci. In Aim 3, we will conduct epigenome-wide association studies on cord blood DNA to enable discovery of new genes linking an adverse maternal milieu with offspring adiposity. Replication of significant DNAm findings from HAPO FUS will be conducted in the Gen3G cohort to ensure reproducibility and rigor of this proposal. Methylation in specific genes may serve as biomarkers when evaluating if interventions are effictive. Discovery of novel biomarkers will enable primordial prevention strategies to curtail the vicious cycle of transgenerational obesity.